Numerous methods exist for genetically engineering mammalian cells. There is great interest in genetically engineering mammalian cells for several reasons including the need to produce large quantities of various polypeptides and the need to correct various genetic defects in the cells. The methods differed dramatically from one another with respect to such factors as efficiency, level of expression of foreign genes, and the efficiency of the entire genetic engineering process.
One method of genetically engineering mammalian cells that has proven to be particularly useful is by means of retroviral vectors. Retrovirus vectors and their uses are described in many publications including Mann, et al., Cell 33:153-159 (1983) and Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984). Retroviral vectors are produced by genetically manipulating retroviruses.
Retroviruses are RNA viruses; that is, the viral genome is RNA. This genomic RNA is, however, reverse transcribed into a DNA copy which is integrated stably and efficiently into the chromosomal DNA of transduced cells. This stably integrated DNA copy is referred to as a provirus and is inherited by daughter cells as any other gene. As shown in FIG. 1, the wild type retroviral genome and the proviral DNA have three Psi genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAS.
Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the TRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). Mulligan, R. C., In: Experimental Manipulation of Gene Expression, M. Inouye (ed), 155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D. and R. C. Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984).
If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Mulligan and coworkers have described retroviral genomes from which these Psi sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome. Mulligan, R. C., In Experimental Manipulation of Gene Expression, M. Inouye (ed), 155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D. and R. C. Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984). Additional details on available retrovirus vectors and their uses can be found in patents and patent publications including European Patent Application EPA 0 178 220, U.S. Pat. No. 4,405,712, Gilboa, Biotechniques 4:504-512 (1986) (which describes the N.sub.2 retroviral vector) The teachings of these patents and publications are incorporated herein by reference.
Retroviral vectors are particularly useful for modifying mammalian cells because of the high efficiency with which the retroviral vectors "infect" target cells and integrate into the target cell genome. Additionally, retroviral vectors are highly useful because the vectors may be based on retroviruses that are capable of infecting mammalian cells from a wide variety of species and tissues.
The ability of retroviral vectors to insert into the genome of mammalian cells have made them particularly promising candidates for use in the genetic therapy of genetic diseases in humans and animals. Genetic therapy typically involves (1) adding new genetic material to patient cell in vivo, or (2) removing patient cells from the body, adding new genetic material to the cells and reintroducing them into the body, i.e., in vitro gene therapy. Discussions of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in U.S. Pat. Nos. 4,868,116, issued Sep. 19, 1989, and 4,980,286, issued Dec. 25, 1990 (epithelial cells), WO89/07136 published Aug. 10, 1989 (hepatocyte cells) , EP 378,576 published Jul. 25, 1990 (fibroblast cells), and WO89/05345 published Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells), the disclosures of which are incorporated herein by reference.
In order to be useful for the various techniques of gene therapy, suitable retroviral vectors require special characteristics that have not hitherto been available. A primary source of the need for these special requirements of the vector for use in the in vivo genetic manipulation of patient cells in gene therapy is because it is usually not feasible to use retroviral vectors that require a selection for integration of the vector into the genome of "patient" cells. For example, typical retroviral vectors, e.g., MSV DHFR-NEO described in Williams, et al., Nature 310:476-480 (1984), uses neomycin resistance as a suitable marker for detecting genetically modified cells. Thus, with such neomycin resistant retroviral vectors, patients would be required to be exposed to high levels of neomycin in order to effect genetic repair of cells through in vivo gene therapy. Moreover, in both in vivo and in vitro gene therapy it may be undesirable to produce the gene product of the marker gene in cells undergoing human gene somatic therapy. For example, there is no therapeutic reason to produce large levels of neomycin phosphotransferase in blood cells undergoing hemoglobin gene replacement for curing a thalassemia. Therefore, it would be desirable to develop retroviral vectors that integrate efficiently into the genome, express desired levels of the gene product of interest, and are produced in high titers without the coproduction or expression of marker products such as antibiotic resistance factors.
Despite considerable progress in efforts to develop effective genetic therapies for diseases involving hematopoietic cells, a number of significant technical hurdles remain. First, while a variety of transduction protocols have been developed which make it possible to efficiently transfer genes into murine hematopoietic stem cells, it has not yet been possible to achieve efficient gene transfer into reconstituting cells of large animals. It is currently unclear to what extent this problem is vector related (e.g. insufficient titers, host range) or a consequence of a lack of knowledge regarding the optimal conditions for obtaining the proliferation and/or efficient engraftment of appropriate target cells. A second important technical stumbling block relates to the development of retroviral vectors possessing the appropriate signals for obtaining high level constitutive expression of inserted genes in hematopoietic cells in vivo. Although a number of groups have demonstrated the expression of genes in mice reconstituted with transduced bone marrow cells, others have experienced difficulties (10-12). Overall, few general principles regarding features of vector design important for gene expression in vivo have emerged. In particular, because of differences in vector backbones, inserted genes, viral titers, transduction protocols, and other experimental parameters, it has been impossible to directly compare the performance of different vectors and to determine the features of vector design which most critically affect gene expression in hematopoietic cells in vivo. In addition, few studies have examined the ability of transferred genes to be expressed for very long periods of time (e.g. the lifetime of the transplant recipients), a clearly important goal of gene therapy for diseases involving hematopoietic cells.